Invasive aspergillosis (IA) caused by Aspergillus fumigatus is among the leading infectious causes of death in immunocompromised patients. Protein kinase A (PKA) signaling is a critical pathway required for the virulence of several important fungal pathogens, including A. fumigatus. However, little is known regarding the specific targets of PKA in any organism. Furthermore, the majority of mechanistic studies relating to the pathway in fungi have been limited to non-pathogenic model yeasts. Unraveling the PKA signaling pathway should therefore provide key insight into the mechanisms of fungal pathogenesis. We have recently identified novel regulatory mechanisms of PKA activity in A. fumigatus via phosphorylation of the catalytic and regulatory subunits. We have also defined the PKA interactome via mass spectroscopic analysis that identified numerous potential target proteins. These include both known and uncharacterized transcriptional regulators, which hint at diverse and novel roles for PKA signaling in this species. These findings underscore the contrast between PKA regulatory mechanisms in filamentous fungi and yeasts, and also signify that important undefined variations exist in the activation of downstream effectors governing virulence and disease establishment. We hypothesize that PKA acts as one master regulator of fungal pathogenic processes through the activation of specific effector proteins that promote growth and virulence. Our overall goal is to identify proteins that significantly contribute to the virulence of A. fumigatus in a PKA-dependent manner. To identify these effectors and better understand the molecular mechanism of A. fumigatus pathogenesis, we will utilize a high-resolution proteomics-based approach to now define the full PKA-dependent phosphoproteome. Following identification, the PKA-dependent effectors will be screened for their contributions to virulence using genetic, in vitro, and animal model strategies. In Aim 1, we will define the A. fumigatus native proteome and PKA-dependent phosphoproteome using two independent quantitative global proteomic approaches. Effectors will be defined as those phosphorylated in a wild-type strain but dephosphorylated in a PKA-deficient mutant strain under basal or stress-inducing conditions. Comparison of specific protein levels between the native proteomes of the wild-type and mutant strains will be used to validate changes in phosphorylation versus changes in protein expression. In Aim 2, we will identify PKA-dependent effectors with virulence contributions. Following prioritization via a bioinformatic and proteomic scoring system, we will use an iterative approach of genetic deletion, in vitro growth screening, and a robust murine model of IA for validation of virulence defects. This strategy will enable the comprehensive, high-resolution identification of PKA-dependent effectors specifically contributing to the growth and virulence of A. fumigatus. Cataloguing the most complete set of PKA targets for any fungal species to date, as well as the identification of specific sites of phosphorylation, will greatly increase our understanding of the fundamental pathobiology of A. fumigatus and potentially facilitate future studies toward novel targeting strategies.